Expression in Escherichia coli of a Chemically
Synthesized Gene for the Hormone
Somatostatin
The chemical synthesis of DNA and
recombinant DNA methods provide
the technology for the design and
synthesis of genes that can be fused to
Plasmid elements for expression in
Escherichia coli or other bacteria. As a
model system we have designed and
Synthesized a gen for the small
polypeptide hormone, Somatostatin.
The major considerations in the choice
on this hormone were its small size
and known amino acid sequence,
sensitive radioinmune and biological
assays, and its intrinsic biological
interest. Somatostatin is a the choice of codons was somewhat
tetradecapeptide; it was originally
discovered in ovine hypothalamic
extracts but subsequently was also
found in significant quantities in other
species and other tissues
Somatostatin inhibits the secretion of a
number of hormones, including growth
hormone, insulin, and glucagón. The
effect of Somatostatin on the secretion
on these hormones has attracted
attention to its potential therapeutic
value in acromegaly, acute
pancreatitis, and insulin-dependent
diabetes. The overall construction of
the Somatostatin gen and Plasmid
was designed to result in the in vivo
synthesis of a precursor form of a
Somatostatin (see Fig. 1). The
precursor protein would not be
expected to have biological activity,
but could be converted to a functional
form by cyanogen bromide cleavage
after celular extraction. The synthetic
Somatostatin gen was fused to the Lac
operon because the controllling sites
of this operon are well characterized.
Given the amino acid sequence of
Somatostatin, one can design form the
genetic code a short DNA fragment
containing the information for its 14
amino acids. The degeneracy of the
code allows for a large number of
posible sequences that could code for
the same 14 amino acids. Therefore,
arbitrary except for the following
restrictions. First, amino acids codons
known to be favored in E. coli for
expression of the MS2 genome were
used were appropiate. Second, since
the complete sequence would be
constructed from a number
overlapping fragments, the fragments
were designed to eliminate
undesirable inter-and intramolecular
pairing. And Third, GC-rich (guanine-
cytocine) followed by AT-rich
(adenine-thymine) sequences were
avoided since they might terminate
transcription. Eight oligonucleotides,
varying in length 11 to 16 nucleotides,
labeled lactosidase subunit Structure
(pSOM11-3). The plasmid
construction escheme (Fig. 3) begins
with plasmid pBR322, a well
characterized cloning vehicle. The Lac
elements were introduced to this
plasmid by insertion of an Hae III
restriction endonuclease fragment
(203 nucleotides) carrying the Lac
promoter, catabolite-gene-activator-
protein binding site, operator,
ribosome binding site, and the First
seven amino codons of the ß-
galactosidase structural gene (fig. 3
and 4).